Pyrimidine compounds and pyrimido indole compounds and methods of use

ABSTRACT

The present invention discloses a compound comprising the formula: 
                         
wherein R is hydrogen or an alkyl group having from one to ten carbon atoms, or a compound of the formula wherein the S is replaced by CH 2 , and optionally comprising a pharmaceutically acceptable salt, hydrate, or solvate thereof. A method of treating a patient having cancer or a disease comprising administering to a patient an effective amount of the compound or pharmaceutically acceptable salt, hydrate, or solvate thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This utility patent application claims the benefit of U.S. ProvisionalPatent Application Ser. No. 62/035,234, filed Aug. 8, 2014. The entirecontents of U.S. Provisional patent Application Ser. No. 62/035,234 isincorporated by reference into this utility patent application as iffully written herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersRO1CA142868, CA136944, CA125153, and CA152316 awarded by the NationalInstitutes of Health, National Cancer Institute, and under grant numberRO1AI098458 awarded by the National Institutes of Health, NationalInstitute of Allergy and Infectious Diseases. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention provides substituted pyrimidine compounds and pyrimidoindole compounds that are useful in treating a patient having cancer.The compounds of this invention are useful as anti-tubulin agents, asdihydrofolate reductase inhibitors, and as single agent combinationchemotherapeutic agents inhibiting VEGFR-2, PDGFR-β, and humanthymidylate synthase (hTS).

2. Description of the Background Art

Microtubules are ever-changing, dynamic, filamentous polymers which makeup one component of the cytoskeleton. FIG. 1 shows representative knownmicrotubule binding anti-tubulin agents. They are composed of α-tubulinand β-tubulin heterodimers, which polymerize to form long, slendermicrotubule polymers. Amongst many other functions, they are involved inmaintaining cell shape, cell signaling and are involved in celldivision. During mitosis the dynamics of microtubules are critical tonormal function of the mitotic spindle. During the interphase the rateof microtubule turnover is 4- to 100-fold slower than that at theanaphase.¹ After the formation of the mitotic spindle, the actions ofmicrotubules help partition the chromosomes into the two differentdaughter cells. Failure of normal microtubule dynamics can lead tomitotic arrest and subsequent cell death.¹

Microtubule binding agents are the most widely used agents utilized incancer chemotherapy.¹⁻³ FIG. 1 shows representative known microtubulebinding anti-tubulin agents. Based on the site of binding onmicrotubules (see FIG. 2), these agents are classified as Vinca sitebinding agents, paclitaxel site binding agents or colchicine sitebinding agents.¹⁻³ Expression of the β-III tubulin isotype is one of themain reasons for the clinical resistance developed towards the use ofvinorelbine as well as taxanes in a range of solid tumors includinglung, ovarian, breast and gastric. In addition, these agents act assubstrates for P-glycoprotein (P-gp), thereby, rendering them lesseffective against tumors that express P-gp due to low intracellularaccumulation.¹⁻³

SUMMARY OF THE INVENTION

A method of treating a patient is provided having a disease comprisingadministering to a patient an effective amount of one or more of thecompounds of this invention. The method includes administering aneffective amount of a salt, hydrate, or solvate of at least one of thecompound(s) of this invention to the patient.

In one embodiment of this invention, a compound is provided comprisingthe formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate thereof.

In yet another embodiment of this invention, a pharmaceuticalcomposition is provided comprising a therapeutically effective amount ofa compound of the formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate thereof.

In yet another embodiment of this invention, a method of treating apatient having cancer or a disease is provided comprising administeringto a patient an effective amount of a compound comprising formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided having cancer or a diseasecomprising administering to a patient an effective amount of a compoundcomprising formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate or solvatethereof, and including administering an effective amount of said salt,hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate or solvatethereof, and including administering an effective amount of said salt,hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate or solvate thereof, andincluding administering an effective amount of said salt, hydrate, orsolvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate or solvate thereof, and including administering an effectiveamount of said salt, hydrate, or solvate of said compound to saidpatient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate or solvate thereof, and including administering an effectiveamount of said salt, hydrate, or solvate of said compound to saidpatient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate or solvate thereof, and includingadministering an effective amount of said salt, hydrate, or solvate ofsaid compound to said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of known microtubule binding anti-tubulinagents.

FIG. 2 shows different binding sites on microtubules.

FIG. 3 shows structures of known dihydrofolate reductase inhibitors.

FIG. 4 shows structures of inhibitors of thymidylate synthase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides substituted pyrimidine compounds andpyrimido indole compounds that are useful in treating a patient havingcancer. The compounds of this invention are useful as anti-tubulinagents, as dihydrofolate reductase inhibitors, and as single agentcombination chemotherapeutic agents inhibiting VEGFR-2, PDGFR-β, andhuman thymidylate synthase (hTS), respectively.

In one embodiment of this invention, a compound is provided comprisingthe formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a compound is providedcomprising the formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate thereof.

In yet another embodiment of this invention, a pharmaceuticalcomposition is provided comprising a therapeutically effective amount ofa compound of the formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising a therapeutically effective amount of a compound ofthe formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate thereof.

In yet another embodiment of this invention, a method of treating apatient having cancer or a disease is provided comprising administeringto a patient an effective amount of a compound comprising formula:

wherein R is selected from one of the following groups consisting of:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided having cancer or a diseasecomprising administering to a patient an effective amount of a compoundcomprising formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate or solvatethereof, and including administering an effective amount of said salt,hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising formula:

wherein R is 2-OCH₃, 3-OCH₃, 4-OCH₃, or 3,4-diOCH₃, and optionallycomprising a pharmaceutically acceptable salt or hydrate or solvatethereof, and including administering an effective amount of said salt,hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R₁ is hydrogen or an alkyl group having from one to ten carbonatoms, R₂ is hydrogen or an alkyl group having from one to ten carbonatoms, R₃ is hydrogen or an alkyl group having from one to ten carbonatoms, and R₄ is hydrogen or an alkyl group having from one to tencarbon atoms, wherein R₁, R₂, R₃, and R₄ are independently selected andmay be the same or different, and optionally comprising apharmaceutically acceptable salt or hydrate or solvate thereof, andincluding administering an effective amount of said salt, hydrate, orsolvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate or solvate thereof, and including administering an effectiveamount of said salt, hydrate, or solvate of said compound to saidpatient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen or an alkyl group having from one to ten carbonatoms, and optionally comprising a pharmaceutically acceptable salt orhydrate or solvate thereof, and including administering an effectiveamount of said salt, hydrate, or solvate of said compound to saidpatient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

and optionally comprising a pharmaceutically acceptable salt or hydrateor solvate thereof, and including administering an effective amount ofsaid salt, hydrate, or solvate of said compound to said patient.

In another embodiment of this invention, a method of treating a patienthaving cancer or a disease is provided comprising administering to apatient an effective amount of a compound comprising the formula:

wherein R is hydrogen, an alkyl group having from one to ten carbonatoms, NH₂, I, or CN, and optionally comprising a pharmaceuticallyacceptable salt or hydrate or solvate thereof, and includingadministering an effective amount of said salt, hydrate, or solvate ofsaid compound to said patient.

A method of treating a patient is provided having a disease comprisingadministering to a patient an effective amount of one or more of thecompounds of this invention. The method includes administering aneffective amount of a salt, hydrate, or solvate of at least one of thecompound(s) of this invention to the patient.

As used herein, the term “effective amount” is defined as the amount ofa compound or composition required to effect a particular result, suchas for example, but not limited to, treating a patient for a disease,including cancer.

As used herein, the term “patient” includes all members of the animalkingdom, including but not limited to, Homo sapiens, warm and coldblooded animals, and reptiles.

The compounds of this application may be administered to a patient inany suitable pharmaceutical form, with or in any suitable pharmaceuticalcarrier, and via a suitable route of administration, including forexample, but not limited to, the oral route, buccal route, rectal route,parenteral route, intraperitoneal route, intramuscular route, ophthalmicroute, dermal route, and inhalation route, to name a few. Apharmaceutically acceptable carrier is any such carrier known to thosepersons skilled in the art and may include for example but is notlimited to saline, dextrose in water, starch, talc, dextrose, orsucrose, and the like.

Section A.

Design, Synthesis and Biological Evaluation of 6-Amino-5-Chloro-2-MethylN⁴-Substituted Pyrimidine Analogs as Anti-Tubulin Agents

Structure of an anti-tubulin compound 1 is set forth below:

TABLE 1 IC₅₀ EC₅₀ Microtubule Compound MDA-MB-435 depolymerization No.(nM) (μM) 1 183 ± 3.4 5.8

TABLE 2 Effect of P-gp on Drug Sensitivity Compound SK-OV-3 SK-OV-3MDR-1-6/6 No. IC₅₀ (nM) IC₅₀ (nM) Rr 1 278 ± 19  435 ± 33 1.6 Paclitaxel 3.0 ± 0.006 2600 ± 270 864 CA4P 4.5 ± 0.2  6.6 ± 1.3 1.5

TABLE 3 Effect of β-III Tubulin on Drug Sensitivity Compound HeLa WTβ-III No. IC₅₀ (nM) IC₅₀ (nM) Rr 1 270 ± 21  186 ± 17  0.7 Paclitaxel1.6 ± 0.2 7.7 ± 0.2 4.7 CA4P 4.7 ± 0.2 5.7 ± 0.4 1.2

Gangjee et al.⁴ had previously reported pyrrolo[2,3-d]pyrimidines asanti-tubulin agents binding to the colchicine binding site. Compound 1was reported to be a microtubule depolymerizing agent, which inhibitedthe growth of cancer cells in the nanomolar range (Table 1). Thiscompound further inhibited β-III expressing and P-gp expressing celllines (Table 2, Table 3).

The present invention provides monocyclic pyrimidine compounds 2-8(Section A.) with the following structures:

wherein R′ is —O—CH₃ or —S—CH₃, and R is H or CH₃.

To further explore the requirements of a bicyclic scaffold it was ofinterest to simplify the structure of compound 1 and determine theminimum structural features that would allow potent cytotoxic andmicrotubule disrupting activities. To this end the monocyclic pyrimidineanalogs derived from the conformationally rigid bicyclic 1 weredesigned. Based on this strategy Gangjee et al.⁵ reported6-chloro-N⁴,2-dimethyl-N⁴-substituted-4,5-diamine of the generalstructure of compound 2 as a single agent with dual actingantiangiogenic and anti-tubulin activity. The compounds presented hereare a continued effort to explore the structure activity relationships(SAR) on the monocyclic pyrimidine core. The effects of interchangingthe chlorine and amine substituents at the C5 and C6, thus, altering theelectronics of the core scaffold were evaluated. Different anilines weresubstituted at C4 to determine the optimum substitution.

Syntheses of Section A. Compounds:

The syntheses of compounds 3-7 set forth above are presented in Scheme1A and Scheme 2A.

Compound 9 (Scheme 1A) was pivaloyl protected using pivalic anhydride togive 10 in 70-98% yield. S_(N)Ar substitutions with various anilines atC4 was then carried out under microwave conditions to give compounds 3(43%), 5 (32%), 6 (26%) and 7 (32%). Compound 4 was synthesized bydeprotecting 13 under basic conditions. N-methylation of anilines 15 and19 was carried out using paraformaldehyde under reductive aminationconditions to yield anilines 14 (39%) and 18 (46%, Scheme 2A).⁶

The structure of Compound 4 using ¹³C NMR is set forth below:

Biological Activity of Section A. Compounds

Compounds 3-7, Section A., were tested for their ability to inhibit cellproliferation and to affect microtubule depolymerization (Table 4). Theywere also tested in P-gp (Table 5) and β-III (Table 6) expressing celllines to assess their ability to overcome resistance.

TABLE 4 IC₅₀ ± SD in EC₅₀ for Microtubule Compound MDA-MB-435Depolymerization EC₅₀/IC₅₀ No. Cells (nM) in A-10 Cells (nM) Ratio 3ND >10 μM  ND 4 71.3 ± 6.1 1.5 μM 21.0 5 135.6 ± 12.5 3.3 μM 24.3 6215.3 ± 23.5 1.4 μM 6.5 6′ 2,076 ± 464   11 μM 5.3 7 in progress ~10 μM — Paclitaxel  4.5 ± 0.52 — — CA-4  4.4 ± 0.46 9.8 2.2 CA-4 =combretastatin A-4; ND = not determined

TABLE 5 Effect of P-gp on Drug Sensitivity Compound SK-OV-3 SK-OV-3MDR-1-6/6 No. IC₅₀ (nM) IC₅₀ (nM) Rr 3 ND ND ND 4 112.1 ± 16.4 180.2 ±32.8 1.6 5 277.1 ± 13.3 414.7 ± 66.2 1.5 6 458.8 ± 22.2 576.0 ± 42.4 1.36′ 3,270 ± 165  2,979 ± 52  0.9 7 in progress in progress Paclitaxel 5.0± .6 1,200 ± 58  240 CA-4  5.5 ± 0.5  7.2 ± 1.1 1.3 Rr = relativeresistance; CA-4 = combretastatin A-4; ND = not determined

TABLE 6 Effect of β-III Tubulin on Drug Sensitivity Compound IC₅₀ ± SDin IC₅₀ ± SD in No. HeLa (nM) HeLa WTβ-III (nM) Rr 3 ND ND ND 4 86.3 ±8.7 111.7 ± 9.2  1.4 5 158.2 ± 10.6 163.3 ± 25.8 1.0 6 312.3 ± 41.0278.6 ± 69.0 0.9 6′ *2,490 ± 90.9  2,573 ± 418  * 1.0   7 in progress inprogress Paclitaxel  2.8 ± .36 24.0 ± 3.0 8.6 CA-4  3.3 ± 0.4  3.3 ± 0.31   * n = 2; Rr = Relative resistance; CA-4 = Combretastatin A-4; ND =Not Determined

Comparing compounds 3 and 4, section A., it can be said that N⁴—CH₃substitution is necessary for activity. Compound 5 with a 4′-SCH₃substitution is two fold less potent than compound 4. Pivaloyl group atN6 is not tolerated well as can be seen by comparing activities ofcompounds 6 and 6′. Amongst all the compounds tested, compound 4 was themost potent compound in this series.

Based on the biological activities, it could be said that thesecompounds possessed two digits to three digits nanomolar values forinhibition of cell proliferation as exemplified by compounds 4-6,Section A. However, these compounds were not a substrate for resistanceby P-gp in that cells lines expressing P-gp were equally sensitive ascompared to the parental cell line. The compounds were additionally ableto overcome resistance due to the expression of β-III tubulin and henceare effective in multidrug resistant cancer cell lines.

SECTION A. REFERENCES

-   1. Dumontet, C.; Jordan, M. A. Microtubule-binding agents: a dynamic    field of cancer therapeutics. Nat. Rev. Drug Discov. 2010, 9,    790-803.-   2. Kavallaris, M. Microtubules and resistance to tubulin-binding    agents. Nat. Rev. Cancer 2010, 10, 194-204.-   3. Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer    drugs. Nat. Rev. Cancer 2004, 4, 253-265.-   4. Gangjee, A.; Zhao, Y.; Lin, L.; Raghavan, S.; Roberts, E. G.;    Risinger, A. L.; Hamel, E.; Mooberry, S. L. Synthesis and discovery    of water-soluble microtubule targeting agents that bind to the    colchicine site on tubulin and circumvent Pgp mediated    resistance. J. Med. Chem. 2010, 53, 8116-8128.-   5. Ganjee, A.; Mohan, R.; Bai, R.; Hamel, E.; Inhat, M. Design,    synthesis and biological evaluation of substituted monocyclic    pyrimidines with dual antiangiogenic and cytotoxic antitubulin    activities as antitumor agents. Abstracts of Papers, 246th ACS    National Meeting, Indianapolis, Ind., United States, Sep. 8-12,    2013.-   6. Teichert, A.; Jantos, K.; Harms, K.; Studer, A. One-pot homolytic    aromatic substitutions/HWE olefinations under microwave conditions    for the formation of a small oxindole library. Org. Lett. 2004, 6,    3477-3480.    Section B.    6-Substituted Pyrrolo[2,3-D]Pyrimidines as Dihydrofolate Reductase    Inhibitors and Potential Anti-Opportunistic Agents

Pneumocystis jirovecii (pj), Toxoplasma gondii, Mycobacterium avium andM. intracellulare are some of the most common organisms that causelife-threatening opportunistic infections in AIDS and otherimmunocompromised patients.¹ Despite the existence of the highly activeantiretroviral therapy (HAART), the incidences of HIV cases persist dueto nonadherence, toxicity arising from current treatments, emergence ofresistant strains, late diagnosis of HIV and a rise in HIV cases indeveloping countries.² Pneumocystis pneumonia (PCP) was originallythought to be caused by fungus Pneumocystis carinii (pc), but it is nowknown that the strain that is responsible for infecting humans is P.jirovecii (pj). P. carinii (pc) is the strain that infects rats.³

Dihydrofolate reductase (DHFR) contributes to the de novo mitochondrialthymidylate biosynthesis pathway. DHFR catalyzes the reduction of7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate using NADPH as reductant.Due to the vital role of DHFR in the folate cycle as well as inthymidylate biosynthesis, the inhibition of DHFR leads to a “thyminelesscell death”.⁴ DHFR enzymes from P. jirovecii and P. carinii (pc) differby 38% in amino acid sequence and exhibit different sensitivity toexisting drugs.⁵ No crystal structure of pjDHFR has been reported todate and known pcDHFR inhibitors act as poor surrogates for pjDHFRinhibition.⁶ In addition, difficulties in in-vitro cultures of P.jirovecii outside of human lung and the lack of animal models haveimpeded the drug discovery efforts to obtain a selective pjDHFRinhibitor.⁷

First line therapy of PCP includes lipophilic, non-classical antifolatesas trimethoprim (TMP) and pyrimethamine (see FIG. 3—structures of knowndihydrofolate reductase inhibitors).⁸ Both TMP and pyrimethamine areweak inhibitors of pjDHFR and must be coadministered with sulfonamidesto compensate for their weak activities.¹ However, combination therapyis successful only in 50-75% of the AIDS population and is limited dueto severe side effects.⁹ Trimetrexate (TMQ) and piritrexim (PTX) (FIG.3) are potent, but non-selective DHFR inhibitors used in the treatmentof moderate to severe PCP. However, they cause high rates ofmyelosuppression and TMQ is coadministered with leucovorin(5-formyltetrahydrofolate) as a rescue agent to prevent host celltoxicity.¹⁰ However, this dual therapy increases treatment cost and hostcell rescue with leucovorin is not always successful. Given thelimitations of the existing regimen, it is highly desirable to developsingle agent DHFR inhibitors that combine the potency of TMQ or PTX withthe species selectivity of TMP and would eliminate the need tocoadminister sulfonamide or leucovorin. The present invention providessuch compounds.

Section B. Compounds:

The present invention provides the following compounds: selective pjDHFRinhibitor compound 1, novel DHFR inhibitor compounds 2-5, Section B.,and their N7-methyl analogs-compounds 6-9, Section B., as set forthbelow:

In 2013, Gangjee et al.¹¹ reported a series of2,4-diamino-5-methyl-6-(arylthio)-thieno[2,3-d]pyrimidines as potent andselective inhibitors of pjDHFR. In this series compound 1 displayed a3-fold higher selectivity against human derived pjDHFR compared toclinically used TMQ. Compound 1 with a 3,4-dimethoxy substitution in theside-chain aryl moiety shows similarity in the side chain substitutionto clinically used TMP, TMQ and PTX. To further explore the structureactivity relationship (SAR) of this series of compounds and to optimizethe potency and selectivity against pjDHFR and other pathogen DHFR, aseries of compounds 2-9 was designed with methoxy group variations inthe aryl moiety of compound 1.

Syntheses of Section B. Compounds:

Intermediate compound 12 (Scheme 1B) was prepared by a 2-step procedurereported by Taylor et al.¹² Acetol 10 was condensed with malononitrilein the presence of triethylamine in methanol to afford2-amino-3-cyano-4-methylfuran (compound 11) which was condensed withguanidine hydrochloride in presence of sodium methoxide to giveintermediate (compound 12) in 44% yield. The synthesis of targetcompounds 2-9, outlined in Scheme 1B, involved oxidative thiolation ofthe common intermediate 2,4-diamino-5-methyl-pyrrolo[2,3-d]pyrimidine(compound 12) with appropriately substituted thiols. Compounds 2-5 weresynthesized from compound 12 with slight modification of the oxidativethiolation previously reported by Gangjee et al.¹³ This procedureinvolved reacting compound 12 with appropriately substituted thiols andiodine in a 2:1 mixture of ethanol and water at reflux to give compounds2-5. Compounds 6-9 were synthesized by methylation of the pyrrolenitrogen using sodium hydride and iodomethane.

Biological Activity of Section B. Compounds:

TABLE 7 Inhibitory concentrations (IC₅₀, in μM) against recombinantpjDHFR, human DHFR (hDHFR) and selectivity ratios ^(a) Compound pjDHFRIC₅₀ hDHFR IC₅₀ Selectivity No. (μM) (μM) hDHFR/pjDHFR 1 0.260 0.910 3.52 0.177 0.624 3.53 3 0.213 0.97 4.55 4 0.252 1.41 5.66 5 3.900 6.9 1.806 0.210 1.4 6.7 7 0.160 1.9 11.9 8 — — — 9 0.234 1.1 4.70 TMP^(b) 0.12032.2 268 TMQ^(b) 0.0021 0.0026 1.2 ^(a) These assays were carried out at37° C. under 9 μM dihydrofolic acid concentration ^(b)These assays werecarried out at 37° C. under 18 μM dihydrofolic acid concentration

Compounds 2-9 of Section B. were evaluated as inhibitors of pjDHFR andhDHFR and the results are reported in Table 7. Selectivity ratios,expressed as IC₅₀ against hDHFR/IC₅₀ against pjDHFR (h/pj) are alsolisted in Table 7. The inhibitory activities of TMP and TMQ are listedfor comparison. While the tested compounds displayed reduced potencyagainst pjDHFR compared to TMQ, they showed 2 to 5 fold greaterselectivity. On comparison of the compounds from the two series ofSection B. compounds, as observed in compound pairs 2 and 6, 3 and 7 and5 and 9, methylation of the pyrrole nitrogen improved the selectivityratios by 2 to 3 fold. Compound 7, the most selective compound in theseries of compounds of this Section B, was about 10-fold more selectivefor pjDHFR over hDHFR than TMQ. None of the compounds synthesizedexhibited potency greater than TMQ, but had greater selectivity comparedto TMQ.

SECTION B. REFERENCES

-   1. Kaplan, J. E.; Benson, C.; Holmes, K. H.; Brooks, J. T.; Pau, A.;    Masur, H. Centers for Disease Control and Prevention (CDC); National    Institutes of Health; HIV Medicine Association of the Infectious    Diseases Society of America: Guidelines for prevention and treatment    of opportunistic infections in HIV-infected adults and adolescents:    recommendations from CDC, the National Institutes of Health, and the    HIV Medicine Association of the Infectious Diseases Society of    America. MMWR Recomm. Rep. 2009, 58, 1-207.-   2. a) Catherinot, E.; Lanternier, F.; Bougnoux, M. E.; Lecuit, M.    Couderc, L. J.; Lortholary, O. Pneumocystis jirovecii pneumonia.    Infect. Dis. Clin. N. Am. 2010, 24, 107-138. b) Ong, E. L. C. Common    AIDS-Associated Opportunistic Infections. Clinical Medicine 2008, 8,    539-543.-   3. Gangjee, A.; Kurup, S.; Namjoshi, O. Dihydrofolate reductase as a    target for chemotherapy in parasites. Curr. Pharm. Des. 2007, 13,    609-639.-   4. MacKenzie, R. E. Biogenesis and interconversion of substituted    tetrahydrofolates. in Folates and Pterins Chemistry and    Biochemistry; Blakley, R. L., Benkovic, S. J., Eds.; Wiley: New    York, 1984; Vol. I, 255-306.-   5. Ma, L.; Kovacs, J. A. Expression and characterization of    recombinant human-derived Pneumocystis carinii dihydrofolate    reductase. Antimicrob. Agents Chemother. 2000, 44, 3092-3096.-   6. Cody, V.; Chisum, K.; Pope, C.; Queener, S. F. Purification and    characterization of human-derived Pneumocystis jirovecii    dihydrofolate reductase expressed in Sf21 insect cells and in    Escherichia coli. Protein Expr. Purif. 2005, 40, 417-423.-   7. Thomas, C. F.; Limper, A. H. Current insights into the biology    and pathogenesis of Pneumocystis Pneumonia. Nat. Rev. Microbio.    2007, 5, 298-308.-   8. Klepser, M. E.; Klepser, T. B. Drug treatment of HIV-related    opportunistic infections. Drugs 1997, 53, 40-73.-   9. Roudier, C.; Caumes, E.; Rogeaux, O.; Bricaire, F.; Gentilini M.    Adverse cutaneous reactions to trimethoprim-sulfamethoxazole in    patients with the acquired immunodeficiency syndrome and    Pneumocystis carinii pneumonia. Arch. Dermatol. 1994, 130,    1383-1386.-   10. Allegra, C. J.; Kovacs, J. A.; Drake, J. C.; Swan, J. C.;    Chabner, B. A.; Masur, H. Activity of antifolates against    Pneumocystis carinii dihydrofolate reductase and identification of a    potent new agent. J. Exp. Med. 1987, 165, 926-931.-   11. Gangjee, A.; Choudhary, S.; Zhou, X.; Queener, S. F.; Cody, V.    Design, synthesis and biological evaluation of substituted    thieno[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and    potential anti-opportunistic agents. Abstracts of Papers, 246th ACS    National Meeting, Indianapolis, Ind., United States, Sep. 8-12, 2013-   12. Taylor, E. C.; Patel, H. H.; Jun, J. G. A one-step ring    transformation/ring annulation approach to    pyrrolo[2,3-d]pyrimidines. A new synthesis of the potent    dihydrofolate reductase inhibitor TNP-351. J. Org. Chem. 1995, 60,    6684-6687.-   13. Gangjee, A.; Devraj, R. D.; McGuire, J. J.; Kisluik, R. L.    5-Arylthiosubstituted    2-amino-4-oxo-6-methyl-pyrrolo[2,3-d]pyrimidine antifolates as    thymidylate synthase inhibitors and antitumor agents. J. Med. Chem.    1995, 38, 4495-4501.    Section C:    Conformationally Restricted Pyrrolo[2,3-d]pyrimidines as Potential    Antimitotic and Antitumor Agents

Disruption of cellular microtubules is a validated target for cancer.¹Three major classes of microtubule active agents (see FIG. 1) have beenidentified according to their binding site on tubulin.² Vinca alkaloidssuch as vincristine, vinblastine consist of the first group which aremicrotubule destabilizers. These are β-tubulin binding agents used inleukemias, lymphomas and other cancers. The second group consist of thetaxoids such as paclitaxel and docetaxel which are designated asmicrotubule stabilizing agents. These agents bind at the interiorsurface of β-subunit of microtubules. They are useful against breast,lung, ovarian and prostate carcinomas. The third group is typified bycolchicine which comprise of a diverse collection of molecules whichbind at the β-tubulin at its interface with α-tubulin. This class ofantimitotic agents is also known as microtubule destabilizers.³Combretastatin A-4 (CA4) and its phosphorylated analog combretastatinA-4 phosphate (CA4-P) which binds to the colchicine site on tubulin iscurrently in clinical trials. There are no approved colchicine sitebinding agents. This demonstrates the importance of developingcolchicine site agents as antitumor agents.^(4,5)

Mutations in the p53 gene occurs in half of all tumors and tubulinbinding agents are highly effective in treating p53 mutant cells.⁶Multidrug resistance (MDR) is a major limitation in cancer chemotherapy,and MDR tumors are resistant to many tubulin-binding agents.⁷Overexpression of P-glycoprotein (Pgp) has also been reported in anumber of tumor types.⁸ Attempts to reverse drug resistance by combiningantimitotic agents with inhibitors of drug efflux proteins produceddisappointing results.³ Expression of βIII-tubulin is another mechanismof resistance to tubulin binding agents in multiple tumor typesincluding non-small cell lung,⁹ breast¹⁰ and ovarian cancer.¹¹ Stengelet al.¹² showed that colchicine site binding agents are the mosteffective agents against βIII-tubulin expressing cells which furtherdemonstrates the importance of developing this class of agents.

Section C. Compounds:

The present invention provides the following compounds 2-9:

In 2010, Gangjee et al.¹³ reported pyrrolo[2,3-d]pyrimidine (compound 1,Section C.) as an inhibitor of the proliferation of human cancer cells(MDA-MB-435). Compound 1, Section C., inhibits the growth of cancercells with GI₅₀ values in the nanomolar range and also circumvents Pgpand βIII-tubulin mediated resistance mechanisms that limit the activityof several microtubule targeting agents.¹³ However, compound 2, SectionC., the N-desmethyl analog of compound 1, is inactive in the cancercells evaluated thus far. The activity of compound 1 was suggested inpart, to involve the 4N-methyl group of compound 1 that aids inmaintaining the relative conformations of the pyrrolo[2,3-d]pyrimidinescaffold and the phenyl ring.¹³ To further extend this finding, a seriesof conformationally restricted analogs, compounds 3-9 of this Section C.were designed based on a systematic approach to restrict theconformation of the phenyl ring relative to the bicyclicpyrrolo[2,3-d]pyrimidine scaffold. A conformational search carried outusing Sybyl 2.1.1 for energy minimized structures of compounds 1,5, and7, Section C., indicated that, compared to compound 1, the number of lowenergy conformations were lowered in compound 5. In compound 7 thenumber of low energy conformations were further lowered. Compound 3,Section C. was designed based on a docking study (not shown) usingLeadIT 2.1¹⁴ which indicated that a potential hydrophobic interactionwith Val181 in the colchicine site of tubulin could be achieved byintroducing a methyl group at the N7 position, which was expected toimprove tubulin inhibitory activity. The docking studied showed astereoview of a superimposition of the docked poses of compound 3 andDAMA colchicine in the colchicine site of tubulin at the interface ofthe α-subunit (magenta) and β-subunit (blue) of tubulin.

Chemistry of Section C. Compounds

The synthesis of target compound 3 (Scheme 1C), started with thesynthesis of a reported method for compound 1.¹³2-Bromo-1,1-diethoxyethane (compound 10) was reacted withethyl-2-cyanoacetate to obtain compound 10 which was cyclized tocompound 12 using acetamidine hydrochloride under basic conditions.Chlorination of compound 12 using POCl₃ provided compound 13 in 80%yield. Displacement of the chloride of compound 13 with4-methoxy-N-methyl aniline (compound 14) and catalytic amounts of HCl inisopropanol, provided compound 1. Methylation of compound 1 with MeIunder basic conditions afforded compound 3 in 85% yield. The synthesisof target compound 5 (Scheme 1C), involved N-formylation of4-methoxy-2-methylanline (compound 15) to afford compound 16 in 70%yield. LAH reduction of compound 16 provided substituted anilinecompound 17. Displacement of the chloride of compound 13 with anilines(compounds 15 and 17) and catalytic amounts of HCl in isopropanolprovided compounds 4 and 5 (75% and 70% respectively).

The synthesis of compound 6, Section C., started from4-amino-3,5-dimethylphenol (compound 18, Scheme 2C). Boc anhydride wasused to protect the amino group of compound 18 to obtain compound 19 in70% yield. Methyl iodide was used to alkylate the free hydroxyl group ofcompound 19, to afford compound 20 in 75% yield. Deprotection usingtrifluoro acetic acid in DCM gave the desired aniline compound 21. Thepyrrole nitrogen of compound 13 was Boc protected to obtain compound 22.Displacement of the chloride of compound 22 with compound 21 under basiccondition furnished compound 23 in 30% yield. Methylation of compound 23with MeI followed by deprotection provided desired compound 6, SectionC.

The synthesis of compound 7-9, section C., of this invention are shownin Scheme 3C. The pyrrole nitrogen of compound 13 was methylated withMeI to obtain compound 25. Displacement of the chloride of compounds 13and 25 with compound 21 under acidic conditions provided the desiredcompounds 7 and 8 in 40-45% yield respectively. Further methylation ofcompound 8 by iodomethane under basic conditions gave compound 9 in 72%yield.

Biological Activity of Section C. Compounds:

TABLE 8 IC₅₀ Values For Inhibition Of Proliferation Of Mda-Mb-435 CellsAnd Effect On Microtubule Polymerization IC₅₀ ± SD EC₅₀ IN A-10 CELLS(MDA-MB-435) (TUBULIN POLYMERIZATION COMPOUND TUMOR CELLS INHIBITORYACTIVITY) 1  183 ± 3.4 NM  5.8 MM 2 >10 MM >40 MM 3 63.2 ± 4.7 NM 1.02MM  4 ND >10 MM 5 ND >10 MM 6 ND >10 MM ND = NOT DETERMINED

Compounds 3-6, Section C., were tested for antiproliferative effectsagainst the mda-mb-435 tumor cell line using sulforhodamine b assay (srbassay). Microtubule disrupting effects of compounds 3-6 were evaluatedin a cell-based phenotypic screen. Compounds 4-6 did not showdepolymerization of microtubules upto 10 μm indicating that compounds4-6 were inactive. Compound 3, Section C., however causeddepolymerization of microtubules and was 5-fold more potent thancompound 1, section C., and inhibited mda-mb-435 tumor cells with a2-fold better IC₅₀ than compound 1. Based on the potent activity ofcompound 3, Section C., compounds 8 and 9 with n7-CH₃ groups weresynthesized along with a n7-H compound 7.

A proton NMR study was carried out, to explore the conformations of 2,3, 5 and 7 which can be considered a representative example of theseries of compounds (compounds 1-9). ¹H NMR spectra for compounds 2, 3,5, and 6 (not shown, having a scale from δ10.0-δ4.0 ppm) in DMSO-d6 wasobtained. The ¹H NMR spectra for compound 2 shows that the sigma bonds(c_(1′)-n and n-c₄) connecting the phenyl ring andpyrrolo[2,3-d]pyrimidine ring are both freely rotatable, while thesebonds are restricted in compounds 3, 5, and 7, where an additionalmethyl group was introduced on the n-4 position. According to ¹ h nmrspectrum (not shown), the 5-h proton in compounds 3, 5, and 7 (δ 4.53,δ4.39 and δ4.37 ppm respectively) are more shielded than compound 2(δ6.55 ppm), which suggests a nearby shielding diamagnetic anisotropiccone. Due to the bulk of the 4-n-methyl group, the conformations ofcompounds 3, 5, and 7 are also restricted such that the phenyl ring hasto position itself on top of the 5-h proton, which leads to the observedshielding effect in compounds 3, 5, and 7.

Compound 3, Section C., in which the pyrrole is n-methylated, showsbetter microtubule depolymerization potency than compound 1, Section C.The role of the n7-methyl group to increase activity may be anadditional hydrophobic interaction with the active site val181(supported by the docking study) that is lacking in compounds 4-6 and isresponsible for the potent activity of compound 3, Section C. Inaddition, the 4n-methyl moiety in compound 3 also plays a role in itspotent activity. Inactivity of compound 5 suggests that theconformationally restricted form of compound 5 may not be the bioactiveconformation.

SECTION C. REFERENCES

-   1. Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer    Drugs. Nat. Rev. Cancer 2004, 4, 253-265.-   2. Jordan, M. A.; Kamath, K. How do Microtubule-Targeted Drugs Work?    An Overview. Curr. Cancer Drug Targets 2007, 7, 730-742.-   3. Dumontet, C; Jordan, M. A. Microtubule-binding agents: A Dynamic    Field of Cancer Therapeutics Nat. Rev. Drug Discov. 2010, 9,    790-803.-   4. Kanthou, C.; Tozer, M. T. Microtubule Depolymerizing Vascular    Disrupting Agents: Novel Therapeutic Agents for Oncology and Other    Pathologies. Int. J. Exp. Pathol. 2009, 90, 284-294.-   5. Carlson, R. O. New Tubulin Targeting Agents Currently in Clinical    Development. Expert Opin. Investig. Drugs 2008, 17, 707-722.-   6. Kavallaris, M. Microtubules and resistance to tubulin-binding    agents. Nat. Rev. Cancer, 2010, 3, 194-204.-   7. Ling, V. Multidrug Resistance: Molecular Mechanisms and Clinical    Relevance. Cancer Chemother. 1997, 40, S3-8.-   8. Chiou, J. F.; Liang, J. A.; Hsu, W. H.; Wang, J. J.; Ho, S. T.;    Kao, A. Comparing the Relationship of Taxol-based Chemotherpay    Response with P-glycoprotein and Lung Resistance-related Protein    Expression in Non-Small Cell Lung Cancer. Lung 2003, 181, 267-273.-   9. Seve, P.; Isaac, S.; Tredan, O.; Souquet, P.-J.; Pacheco, Y.;    Perol, M.; Lafanechere, L.; Penet, A.; Peiller, E.-L.; Dumontet, C.    Expression of Class III β-Tubulin Is Predictive of Patient Outcome    in Patients with Non-Small Cell Lung Cancer Receiving    Vinorelbine-Based Chemotherapy. Clin. Cancer Res. 2005, 11,    5481-5486.-   10. Tommasi, S.; Mangia, A.; Lacalamita, R.; Bellizzi, A.; Fedele,    V.; Chiriatti, A.; Thomssen, C.; Kendzierski, N.; Latorre, A.;    Lorusso, V.; Schittulli, F.; Zito, F.; Kavallaris, M.; Paradiso, A.    Cytoskeleton and Paclitaxel Sensitivity In Breast Cancer: The Role    Of Beta-Tubulins. Int. J. Cancer 2007, 120, 2078-2085.-   11. Ferrandina, G.; Zannoni, G. F.; Martinelli, E.; Paglia, A.;    Gallotta, V.; Mozzetti, S.; Scambia, G.; Ferlini, C. Class III    β-Tubulin Overexpression Is A Marker Of Poor Clinical Outcome In    Advanced Ovarian Cancer Patients. Clin. Cancer Res. 2006, 12,    2774-2779.-   12. Stengel, C; Newman, S. P.; Lesse, M. P.; Potter, B. V. L.;    Reed, M. J.; Purohit, A. Class III Beta-Tubulin Expression and in    vitro Resistance To Microtubule Targeting Agents. Br. J. Cancer    2010, 102, 316-324.-   13. Gangjee, A.; Zhao, Y; Lin, L.; Raghavan, S.; Roberts, E. G.;    Risinger, A. L.; Hamel, E.; Mooberry S. L. Synthesis and Discovery    of Water-Soluble Microtubule Targeting Agents that Bind to the    Colchicine Site on Tubulin and Circumvent Pgp Mediated    Resistance. J. Med. Chem. 2010, 53, 8116-8128.-   14. LeadIT, version 2.1; BioSolveIT GmbH: Sankt Augustin, Germany.-   15. Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar,    S.; Sobel, A.; Knossow, M. Nature, 2004, 428, 198-202.    Section D:    5-Substituted Pyrimido[4,5-b]indoles with Single Agent Combination    Chemotherapeutic Potential

Thymidylate synthase (TS) converts dUMP (deoxyuridine monophosphate) todTMP (deoxythymidine monophosphate) by transferring a methyl group viathe cofactor 5,10-methylenetetrahydrofolate. This is an important stepfor DNA synthesis and cell growth, thus TS is a viable target forseveral clinically used cancer chemotherapeutic agents.^(1,2) Thefluoropyrimidine, 5-fluorouracil (5-FU) and its derivatives, inparticular, capecitabine (FIG. 4), have found extensive utility inovarian, breast, colon, and several other cancers alone and incombinations and are a mainstay in cancer chemotherapy.³ Folateinhibitors of TS, pemetrexed (PMX)⁴ and in Europe raltitrexed (RTX)⁵,are used alone or in combination in the clinic against a variety ofcancers (see FIG. 4 structures).

Angiogenesis is the process of formation of new blood vessels fromexisting vasculature—is essential for tumor growth and metastasis.⁶Receptor tyrosine kinases (RTKs) play a crucial role in angiogenesis.RTKs are enzymes that catalyze the transfer of the γ-phosphate of ATP totyrosine residues of protein substrates. Vascular endothelial growthfactor receptor (VEGFR), epidermal growth factor receptor (EGFR) andplatelet derived growth factor receptor (PDGFR) are common RTKs that areoverexpressed in cancer cells.⁷ Agents that block angiogenesis byinhibition of RTKs have established a new paradigm in cancerchemotherapy. Single RTK inhibitors are prone to resistance by numerousmechanisms including point mutations in the ATP binding site andupregulation of additional RTKs.⁸ Consequently multi-RTK inhibition incancer chemotherapy has emerged as a promising approach and its validityhas been highlighted by the approval of several multi-RTK inhibitorsincluding sorafenib [inhibits VEGFR-2, PDGFR-β, Flt-3 (FMS-like tyrosinekinase-3), Raf kinase and c-kit]⁹ and sunitinib [inhibits VEGFR-1,-2,and -3, PDGFR-β, -α, stem cell factor (kit), Flt-3 and CSF-1R(colony-stimulating factor-1 receptor)] (FIG. 1).¹⁰

Combination cancer chemotherapy is not a novel concept. Recent studiessuggest that the combination of separate cytostatic, antiangiogenicagents with separate cytotoxic agents is more effective in cancerchemotherapy than either agent alone.¹¹ We envisioned the design ofsingle agents that would function by both a cytostatic (antiangiogenic)mechanism and a cytotoxic (antifolate) mechanism. Such single agentswould circumvent the pharmacokinetic problems of two or more agents andreduce drug-drug interactions. In addition, the same agents could beused at lower doses to alleviate toxicity, be devoid of overlappingtoxicities, and delay or prevent tumor cell resistance. Mostsignificantly, providing the cytotoxic agent, by structural design, inthe same molecule allows the cytotoxicity to be manifested as soon asthe antiangiogenic effects are operable.

A separately dosed cytotoxic agent may miss the timing window and hencethwart the intent of the combination. Such multi-targeted agents couldwield their cytotoxic action as soon as or even during transient tumorvasculature normalization^(9, 10) due to the antiangiogenic effects.Thus such agents, perhaps, do not need to be as potent as conventional,separately dosed cytotoxic agents. Other advantages of such singleagents are in the decreased cost and increased patient compliance whichare sometimes as important contributors to chemotherapy failure asresistance, toxicity and lack of efficacy. One of the most importantproblems with conventional cytotoxic chemotherapeutic agents is doselimiting toxicities. These single agents should avoid these toxicitiesas they do not need to be as potent as conventional chemotherapeuticagents.

Compounds 1 and 2, Section D., (see FIG. 4, bottom row, far rightcolumn) each inhibit VEGFR-2 and PDGFR-β for antiangiogenic effects andalso inhibit human TS (hTS) for cytotoxic effects in single agents.¹²The inhibitory potency of both these single agents against VEGFR-2,PDGFR-β, and hTS was better than or close to standards (Tables 9, 10).In a COLO-205 xenograft mouse model, one of the analogs significantlydecreased tumor growth (tumor growth inhibition (TGI)=76% at 35 mg/kg),liver metastases, and tumor blood vessels compared with a standard drugand with control and thus demonstrated potent tumor growth inhibition,inhibition of metastasis, and antiangiogenic effects in vivo. Thesecompounds afford combination chemotherapeutic potential in singleagents.

TABLE 9 IC₅₀ values (μM) of kinase inhibition and A431 cytotoxicity¹²compounds 1-2, Section D. VEGFR-2 VEGFR-1 EGFR (Flk-1) (Flt-1) PDGFR-βKinase Kinase kinase Kinase A431 Compd # Inhibition InhibitionInhibition Inhibition Cytotoxicity 1 15.07 ± 3.1 22.6 ± 4.5 118.1 ± 19.4 2.8 ± 0.42 49.2 ± 4.7 2 10.41 ± 1.2 56.3 ± 7.1 160.1 ± 28.9 40.3 ± 5.1 14.1 ± 2.0 PD153035  0.23 ± 0.05 SU5416 12.9 ± 2.9 CB67645 14.1 ± 2.8DMBI 3.75 ± 0.31 Cisplatin 10.6 ± 3.5

TABLE 10 IC₅₀ Values (μM) of Thymidylate Synthase Inhibition¹² compounds1-2, Section D. Compd # Human E. coli Toxoplasma gondii 1 0.54 >27 0.112 0.39 >26 >26 Pemetrexed 29.0 15 14 Raltitrexed 0.29 2.3 0.48

The present invention provides the compounds of the following formula:

Lead compounds 1 and 2, Section D., showed good activity against VEGFR-2and PDGFR-β in in-cell kinase assays (Table 9). Compounds 1 and 2,Section D., have a sulfur atom as a linker for 5-position substitution.From molecular modeling of the lead compounds 1 and 2, section D., weobserved that the 5-position phenyl ring is surrounded by hydrophobicamino acid residues in VEGFR-2, PDGFR-β and hTS binding sites (notshown). Thus we designed isomers of compounds 1 and 2 with carbon(compounds 3 and 4, Section D.) and oxygen (compound 5, Section D.) atomlinkers in place of the sulfur atom. With the use of calculations usingMOE 2013.08)¹³, these modifications result in changes in the dihedralangle and bond lengths of the linker region, which could alter theorientation of the 5-aryl moiety relative to the tricyclic scaffold.This could provide improved interactions with hydrophobic residues inthe binding site of the targeted kinases and hTS and could result inincreased potency and/or selectivity against RTKs and/or hTS. Dockingstudies of compounds 2 and 5, Section D. (not shown) having astereoview, superimposition of docked poses of compound 2 and compound 5in the ATP binding site of VEGFR-2 (PDB: 1YWN)¹⁴, and stereoview,superimposition of docked poses of compounds 2 and 5, Section D. in thehTS crystal structure were carried out. (PDB: 1JU6)¹⁴

Molecular modeling studies were carried out for compounds 3-5 usingcrystal structures of VEGFR2 (PDB: 1YWN) and hTS (PDB: 1JU6) usingLeadIT 2.1.3. The docking poses were visualized using CCP4MG.¹⁴ Thedocked structure of compound 5 retains interactions at VEGFR-2 bindingsite predicted for the lead compound 2. Both compounds 2 and 5 showhydrogen bonds with Hinge region amino acids (4-NH₂ group with Glu915(C═O); N3 with Cys917 (N—H) and 2NH₂ with Cys917 (C═O)). The 5-phenoxyring fits in hydrophobic pocket 1 and interacts with Val897, Leu1033 andCys1043. As expected, docking studies predict variations in theorientation of the 5-phenoxy ring of compound 5 compared to thecorresponding thiophenyl ring of compound 2 due to the alteredheteroatom linker.

Compound 5, Section D., was predicted (score: −22.84 kJ/mol) to retainthe activity of compound 2 (score: −20.66 kJ/mol) against VEGFR-2.Docking studies for compounds 2 and 5 in hTS show hydrogen bonds betweenthe 2-NH₂ group and the side chain OH of Tyr258, 4NH₂ group and Asp218,and between the pyrrole NH and the backbone carbonyl of Ala111. Dockingalso reveals that the C-ring of both compounds show hydrophobicinteractions with Trp109. In addition, the 5-phenoxy ring makeshydrophobic interactions with Ile108, Leu221, and Phe225 and is involvedin stacking interactions with dUMP in the binding site. Docking studiessuggest that compound 5 should be a potent inhibitor of hTS, similar tolead compound 2. Compounds 3 and 4 display similar docked poses in thetargeted kinases and in hTS (not shown). Therefore, these compounds arepredicted to be potent single agents with multiple RTK and hTSinhibitory activities.

Chemistry of Compounds of Section D.

Suzuki coupling reactions between different trifluoroborate salts(compounds 10 and 13) (Scheme 1D and 2D) with common intermediate2-amino-3-cyano-4-bromo indole (compound 9, Section D.) affordedcompounds 11 and 14 in 31% and 27% yield, respectively. Subsequentcyclization of compounds 11 and 14 with carbamimidic chloridehydrochloride in DMSO₂ at 110° C. gave target compounds 3 and 4 in 20%and 15% yield, respectively. Trifluoroborate salt (compound 10) wascommercially available whereas compound 13 was synthesized using areported procedure.¹⁵ Ullmann type coupling of sodium phenoxide with5-bromo-9H-pyrimido[4,5-b]indole-2,4-diamine (compound 16) (Scheme 3D)afforded target compound 5, Section D., in 19% yield. Intermediatecompound 9 was prepared by a similar synthetic method as described inScheme 1D starting with 2-fluoro-3-bromo nitrobenzene (compound 15)which gave compound 9 in 71% overall yield. Cyclization withcarbamimidic chloride hydrochloride in DMSO₂ at 110° C. gaveintermediate compound 16 in 42% yield.

SECTION D. REFERENCES

-   1) Chu, E.; Callender, M. A.; Farrell, M. P.; Schmitz, J. C. Cancer    Chemother. Pharmacol. 2003, 80, 897.-   2) Danenberg, P. V. Biochim. Biophys. Acta 1977, 47, 73-92.-   3) Pizzorno, G. D., R. B.; Cheng, Y-C. Pyrimidine and Purine    Antimetabolites, in Cancer Medicine, J. F. Holland and E. Frei III,    Eds. B. C. Decker, Inc., Hamilton, London. 2003, 739-744.-   4) Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G.    B.; Barredo, J.; Jannatipour, M.; Moran, R. J. Med. Chem. 1992, 35,    4450-4454.-   5) Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,    M.; Calvert, A. H.; Judson, I. R.; Hughes, L. R. Cancer Res. 1991,    51, 5579-5586.-   6) Carmeliet, P. Nat. Med. 2003, 9, 653.-   7) Herbst, R. S.; Johnson, D. H.; Mininberg, E.; Carbone, D. P.;    Henderson, T.; Kim, E. S.; Blumenschein, G., Jr.; Lee, J. J.;    Liu, D. D.; Truong, M. T.; Hong, W. K.; Tran, H.; Tsao, A.; Xie, D.;    Ramies, D. A.; Mass, R.; Seshagiri, S.; Eberhard, D. A.; Kelley, S.    K.; Sandler, A. J. Clin. Oncol. 2005, 23, 2544.-   8) Hammerman, P. S.; Janneand, P. A.; Johnson, B. E. Clin. Cancer    Res. 2009, 15, 7502.-   9) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.;    Rong, H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.;    Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane,    L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky,    A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res.    2004, 64, 7099.-   10) Mandel, D. B.; Laird, A. D.; Xin, X.; Louie, S. G.;    Christensen, J. G.; Li, G.; Schreck, R. E.; Abrams, T. J.; Ngai, T.    J.; Lee, L. B.; Murray, L. J.; Carver, J.; Chan, E.; Moss, K. G.;    Haznedar, J. O.; Sukbuntherng, J.; Blake, R. A.; Sun, L.; Tang, C.;    Miller, T.; Shirazian, S.; McMahon, G.; Cherrington, J. M. Clin.    Cancer Res. 2003, 9, 327.-   11) Klement, G.; Baruchel, S.; Rak, J.; Man, S.; Clark, K.;    Hicklin, D. J.; Bohlen, P.; Kerbel, R. S. J. Clin. Investig. 2000,    105, R15-R24.-   12) Gangjee, A.; Zaware, N.; Raghavan, S.; Ihnat, M.; Shenoy, S.;    Kisliuk, R. L. J. Med. Chem. 2010, 53, 1563-1578.-   13) Molecular Operating Environment (MOE 2011.10), Chemical    Computing Group, Inc., 1255 University Street, Suite 1600, Montreal,    Quebec, Canada, H3B 3X3. www.chemcomp.com-   14) McNicholas, S.; Potterton, E.; Wison, K. S.; Noble, E. M. Acta    Cryst. 2011, D67, 386-394.-   15) Molander, G. A.; Petrillo, D. E. Org. Synth. 2007, 84, 317-324.    Section E.    Design, Synthesis and Biological Evaluation of Substituted    Monocyclic Pyrimidines with Cytotoxic and Antitubulin Activities as    Antitumor Agents

Cellular microtubules are dynamic filamentous polymers composed of α andβ-tubulin heterodimers, and they play a key role in cell mitosis byforming the mitotic spindle. Microtubule inhibitors disrupt or suppressboth the microtubule structure and its normal functions by inhibition orpromotion of microtubule assembly, resulting in cell cycle arrest in themitotic phase and induction of apoptosis.¹ An overly simplisticclassification of antimitotics includes microtubule-stabilizing orpolymerizing agents (exemplified by taxanes) and microtubuledestabilizing agents (exemplified by the vinca alkaloids).² Taxanes bindto the interior of the microtubule on the β-subunits. In contrast, thevinca alkaloids also bind tubulin but at a site distinct from that oftaxoids. Recently, a prodrug (combretastatin A-4 phosphate, CA4P) of thepotent colchicine site compound combretastatin A-4 (CA4) has beenapproved for clinical trials.³ The colchicine site is primarily onβ-tubulin, but at its interface with the α-subunit of the same tubulinheterodimer. Interfering with microtubule polymerization has been aviable strategy for the development of highly successful antitumor drugclasses. FIG. 1 show the structures of microtubule-targeting agents.

Multidrug resistance (MDR) is a major limitation of clinically usedantimitotic agents, and MDR tumors are usually resistant to microtubuledisrupting agents. Overexpression of P-glycoprotein (Pgp) has beenreported in the clinical setting in several tumor types, particularlyafter patients have received chemotherapy.⁴ Moreover, Pgp expression mayact as a prognostic indicator in certain cancers and is associated withpoor response to chemotherapy by inducing resistance in the presence ofcytotoxic drugs.⁵ Another clinical mechanism of resistance totubulin-binding drugs is the overexpression of specific isotypes ofβ-tubulin, particularly βIII-tubulin. The overexpression of βIII-tubulinin multiple tumor types, including breast, ovarian and non-small celllung cancers,⁶ is involved in resistance to taxanes and vinca alkaloids.

Section E. Compounds:

The present invention provides the following compounds:

This invention provides lead compound 2 and target compounds 3-8,Section E.

Recently, Gangjee et al.⁷ reported a novel compound 1.HCl (Section E,structure shown above) that inhibits tubulin assembly and affordscytotoxic effects. Compound 2, Section E, structure shown above) an openchain conformationally flexible analog of compound 1.HCl, was designedand found to be 5-times more potent against tubulin polymerization ascompared with the lead compound 1.HCl.⁸ This finding prompted a SARstudy and this report addresses the effect of removal of the 6-Cl moietyof compound 2 (see compound 3, Section E, structure shown above) andsubstitution of the 6-Cl moiety of compound 2 with electron donating(compounds 4-6, Section E, structures shown above) and electronwithdrawing groups (compounds 7=8, Section E., structures shown above)on biological activity.

Chemistry of Compounds of Section E.

Dichloropyrimidine (compound 9, Scheme 1E) was subjected to nucleophilicdisplacement with 4-methoxy-N-methylaniline and a catalytic amount ofconcentrated HCl in the presence of i-PrOH to afford compound 2, SectionE. Compound 2, section E., was hydrogenated under Pd/C at 50 psi for 3hours (h) to afford compound 3, Section E. The synthesis of compounds 4and 5, Section E., used trialkylaluminium in the presence of Pd catalystand THF under reflux conditions. Compound 7, Section E., was obtainedfrom 2 using aqueous hydriodic acid at 0° C.-rt. Compound 7 was thenheated at 120° C. in the presence of DMF and copper cyanide to yieldcompound 8, Section E. One of the chloro groups of compound 9, SectionE, was substituted with the amino group under S_(N)Ar conditions withethanolic ammonia to produce compound 10, Section E., which was thensubjected to nucleophilic displacement with 4-methoxy-N-methylanilineand a catalytic amount of concentrated HCl in the presence of butanol toafford compound 6, Section E.

Biological Data for Compounds of Section E.

TABLE 11 Inhibition of tubulin assembly Compd. IC₅₀ ± SD (μM) CA4 0.96 ±0.07  1.HCl 10 ± 0.6  2 2.1 ± 0.04 3 11 ± 0  4 18 ± 2  5 >20 6 >20 7 3.4± 0.07 8 >20 (partial activity)

Table 11 shows that the removal of the 6-chloro (compound 3, section E.)led to a 5.5-fold decrease in potency as an inhibitor of tubulinpolymerization. Replacement of the chloro with lipophilic electrondonating groups (compounds 4 and 5, section E.) led to a furtherdecrease in activity. Compound 5, Section E., with an ethyl at the6-position was inactive. Compound 6, Section E., with a NH₂ at the6-position was also inactive. Substitution of the 6-chloro with an iodomoeity (compound 7, Section E.) was tolerated, albeit with a 1.5-foldreduction in potency. Compound 8, Section E., with a hydrophilicelectron withdrawing nitrile group at the 6-position had partialactivity. Thus, a lipophilic electron withdrawing group at the6-position appears to be necessary for activity.

TABLE 12 Compounds 2, 5, and 7, Section E., Circumvent Pgp MediatedResistance Effect of Pgp on drug sensitivity^(a) IC₅₀ ± SD (nM) ParentalPgp Overexpressing Compd. OVCAR-8 NCl/ADR-RES Rr^(b) paclitaxel 10.0 ±0  5,000 ± 0   500 CA4   6 ± 0.7 9.0 ± 2  1.5 2 25 ± 5 20 ± 0 0.8 5 2200± 500 610 ± 20 0.27 7 450 ± 70 250 ± 70 0.56

TABLE 13 Compounds 3 and 4, Section E., Circumvent βIII-TubulinResistance Effect of βIII on drug sensitivity^(a) IC₅₀ ± SD (nM) Compd.Wild type HeLa βIII Overexpressing HeLa Rr^(b) paclitaxel 5.3 ± 2   16 ±1 3.01 CA4  1.8 ± 0.4   2.5 ± 0.7 1.38 3 1700 ± 500 2500 ± 0 1.47 4 1600± 300 2500 ± 0 1.56 ^(a)Antiproliferative effects of compounds 2-5, 7,Section E., in parental and MDR-1 cell lines in comparison with othermicrotubule disrupting agents. The IC₅₀ values were determined using theSRB assay (n = 3 (SD). ^(b)Rr: Relative resistance. The Rr wascalculated by dividing the IC₅₀ of the Pgp or β-III overexpressing cellline by the IC₅₀ of the parental cell line.

The ability of compounds 5 and 7, Section E., to circumvent Pgp-mediateddrug resistance was evaluated using an OVCAR-8 isogenic cell line pair(Table 12). In this cell line pair, the relative resistance (Rr) ofpaclitaxel is 500 while Rr values of less than 1 were obtained withcompounds 5 and 7, Section E., consistent with the Rr value obtainedwith CA4 of 1.5. Remarkably, compounds 5 and 7, Section E., are1.8-3.6-fold more potent in the Pgp overexpressing cell as compared withthe parental line, indicating a possible utility against paclitaxelresistant ovarian cancer. These data suggest that compounds 2, 5, and 7,Section E., are poor substrates for transport by Pgp and thus haveadvantages over some clinically useful tubulin-targeting drugs likepaclitaxel. A HeLa cell line pair was used to study the effects of βIIItubulin on the potency of compounds 3 and 4, Section E. (Table 13). TheWT βIII cell line was generated from HeLa cells transfected with thegene for βIII-tubulin. Compounds 3 and 4 have Rr values from 1.47-1.56,suggesting that these compounds overcome drug resistance mediated byβIII-tubulin as compared with paclitaxel, which has a Rr of 3.01 inthese cell lines. Thus compounds 2-5, and 7, Section E., inhibit theproliferation of human cancer cells without regard to their expressionof Pgp or βIII-tubulin.

It will be appreciated by those persons skilled in the art that thisinvention provides for the synthesis and evaluation of compounds 2-8,Section E., as tubulin inhibitors and as antitumor agents. It was foundthat the chloro group (or other lipophilic electron withdrawing groupslike iodo) at the 6-position is required for activity against tubulinpolymerization. In addition, compounds 2, 5, and 7, Section E.,displayed better potency in a Pgp overexpressing tumor cell line ascompared with its isogenic control, indicating that these analogsperhaps antagonize Pgp. Compounds 3 and 4, Section E., overcameresistance mediated by βIII-tubulin.

SECTION E. REFERENCES

-   1. Dumontet, C.; Jordan, M. A. Microtubule-binding agents: A dynamic    field of cancer therapeutics. Nat. Rev. Drug Discov. 2010, 9,    790-803.-   2. Jordan, M. A.; Kamath, K. How do Microtubule-targeted drugs work?    An overview. Curr. Cancer Drug Targets 2007, 7, 730-742.-   3. Massarotti, A.; Coluccia, A.; Silvestri, R.; Sorba, G.;    Brancale, A. The Tubulin Colchicine Domain: a Molecular Modeling    Perspective. ChemMedChem. 2012, 7, 33-42.-   4. Fojo, A. T.; Menefee, M. Microtubule targeting agents: Basic    mechanisms of multidrug resistance (MDR). Semin. Oncol. 2005, 32,    S3-S8-   5. McCarron, J. A.; Gan, P. P.; Liu, M.; Kavallaris, M. βIII-Tubulin    is a multifunctional protein involved in drug sensitivity and    tumorigenesis in non-small cell lung cancer. Cancer Res. 2010, 70,    4995-5003.-   6. Chiou, J. F.; Liang, J. A.; Hsu, W. H.; Wang, J. J.; Ho, S. T.;    Kao, A. Comparing the relationship of taxol-based chemotherapy    response with P-glycoprotein and lung resistance-related protein    expression in non-small cell lung cancer. Lung 2003, 181, 267-273.-   7. Gangjee, A.; Pavana, R. K.; Li, W.; Hamel, E.; Westbrook, C.;    Mooberry, S. L. Novel water-soluble substituted    pyrrolo[3,2-d]pyrimidines: design, synthesis and biological    evaluation as antitubulin antitumor agents. Pharm. Res. 2012, 29,    3033-3039.-   8. Gangjee, A.; Mohan R.; Bai R.; Hamel, E.; Ihnat M. From Abstracts    of Papers, 246th American Chemical Society National Meeting and    Exposition (ACS), Indianapolis, Ind., Sep. 8-12, 2011. Abstract No:    311.

It will be appreciated by those persons skilled in the art that changescould be made to the embodiments described above without departing fromthe broad inventive concept thereof. It is understood therefore, thatthis invention is not limited to the particular embodiments disclosed,but it is intended to cover modifications that are within the spirit andscope of the invention, as defined by the appended claims.

What is claimed is:
 1. A compound of the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.
 2. A compound of the formula:

wherein R is an alkyl group having from one to ten carbon atoms, NH₂, I,or CN, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.
 3. A pharmaceutical composition comprising atherapeutically effective amount of a compound of the formula:

and optionally comprising a pharmaceutically acceptable salt or hydratethereof.
 4. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of the formula:

wherein R is an alkyl group having from one to ten carbon atoms, NH₂, I,or CN, and optionally comprising a pharmaceutically acceptable salt orhydrate thereof.
 5. The pharmaceutical composition of claims 3 or 4comprising at least one pharmaceutical carrier.